Regulation by ncRNAs

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(1)

Regulation by ncRNAs

structural RNAs

sRNAs

siRNA miRNA

lncRNAs

pervasive

transcripts

(2)

HISTORY OF RNA

Rinn and Chang, Ann. Rev. Biochem, 2012

(3)

RNA made via condensation from ribose and other organic substances

RNA evolution- molecules

learns to replicate RNA starts to join aminoacids and synthesises polypeptides and proteins

Proteins aid RNA to replicate and make

proteins. dsRNA evolves into stable DNA. DNA and proteins take over major roles as genetic information and enzymes

„primordial soup”

„prebiotic soup”

„THE RNA WORLD” hypothesis

pre-RNA world RNA world

RNA+proteins

RNA+DNA+

proteins

(4)

Library of randomized RNA sequences (1015)

SELEX cycles

1. binding

2. washing 3. elution

4. Amplification

RT-PCR

5. in vitro transcription

final molecules:

cloning, analysis

last cycle

Enriched library

discard -

molecules that do not bind

molecules that bind

Enrichment

Target

tests

SELEX = Systematic Evolution of Ligands by EXponential enrichment

Szostak Gold

1990 Method of selecting RNA/DNA molecules with

desired properties (aptamers, ribozymes) based on cycles of amplification

Selected RNAs:

- cleave DNA i RNA - ligate RNAs

- self-replicate

- create peptide bonds

(5)

ncRNA

Housekeeping

- constitutively expressed

- required for normal function and cell viability

• Regulatory

- expressed temporarily (development, response to stimuli)

- affect gene expression at the level of transcription or translation

tRNA and rRNA – translation

snRNA – splicesosome components, pre-mRNA splicing

snoRNA – rRNA processing and modification, scaRNA (CB specific)

• RNA components of RNase P and RNase MRP – endonucleases:

tRNA and rRNA processing

• Signal Recognition Particle SRP RNA – protein secretion to ER

tmRNA tRNA-mRNA hybrid- targeting nascent proteins for degradation

gRNA – guide RNA in RNA editing

telomerase RNA – synthesis of telomers

sRNAs: siRNA (exo-siRNAs and endo-siRNAs; ta-siRNA; nat-siRNA;

lsiRNAs); miRNA ; piRNA – act in TGS or PTGS

lncRNAs – much less known, usually act in TGS (chromatin level)

(6)

Tisseur et al., Biochemie, 2011

ncRNAs? ALL

(7)

GENE SILENCING - RNAi

DISCOVERY OF 2002:

ncRNAs in RNAi

siRNAs/miRNAs:

- double stranded small noncoding RNAs

- complementary to mRNA targets

- participate in gene silencing

- mediate:

TRANSCRIPTIONAL GENE SILENCING (TGS)

transcription inhibition POST-TRANSCRIPTIONAL GENE SILENCING (PTGS)

mRNA cleavage or

translation inhibition or

translation activation

(8)

Rusk, Nature Meth, 2008

Activation of translation (PTGS)

Interfering RNAs: mode of action

Tijsterman and Plasterk, Cell, 2004

miRNA siRNA

siRNA/miRNA-dependent cleavage (PTGS)

Inhibition of translation (PTGS)

Transcriptional silencing (TGS)

B

artel, Cell, 2004

(9)

PTGS : Post Transcriptional Gene Silencing (RISC)

mRNA cleavage or mRNA decay, translation repression /activation

• siRNA (exo-siRNAs and endo-siRNAs; ta-siRNA; nat-siRNA; lsiRNAs)

• miRNA (mirtron)

• piRNA

TGS : Transcriptional Gene Silencing (RITS)

heterochromatin formation, DNA methylation, transposon silencing

• siRNA

• piRNAs

Holo-RISC

RNA Pol RNA Pol IV

(10)

PTGS : Post Transcriptional Gene Silencing (RISC)

mRNA cleavage or mRNA decay, translation repression/activation

siRNA, miRNA- ( plants, algae, animals, viruses, protists ) ; piRNA (metazoans)

exo-siRNAs - exogenous siRNA, act usually in cis (animals, plants, fungi) viral-siRNAs - exogenous siRNAs, viral genome (plants)

transgene siRNAs - against foreign RNA (animals, plants, fungi)

endo-siRNAs - endogenous siRNAs, act usually in cis

ta-siRNA - trans-acting, generated by miRNA cleavage of ncRNAs (plants)

nat-siRNA- natural antisense-derived siRNAs (plants)

• lsiRNAs - long siRNAs (30-40 nts) derived from antisense RNAs (plants)

mirtron- intron-encoded miRNAs that bypass Dicer processing (animal)

(11)

miRNA

(microRNA)

plants animals viruses, Protista

20–25nt Drosha

(animals)

+ Dicer Pol II /Pol III transcripts mRNA stability

translation inhibition (mRNA cleavage or decay)

mirtrons – derived from pre-mRNA introns; present in animals

siRNA

(small interferring RNA) – most act in cis, except tasiRNA

exo-siRNA

(exogenous)

plants fungi animals Protista

21-24nt Dicer transgenic, virus-derived

or other exogenic RNA PTGS

anti-virus defence

endo-siRNA

(endogenous)

plants fungi, animals Protista

~21nt Dicer

Bi-directional or

convergent transcription mRNA base-pairing with antisense pseudogenes

PTGS,

transposon regulation

tasiRNA

(trans-acting

siRNA) plants 21nt DCL4 TAS RNA precursors

cleaved by miRNA PTGS

natsiRNA

(natural antisense transcripts- derived siRNA)

plants 24nt

21nt DCL2

DCL1 Stress-induced

bi-directional transcription Stress response

sRNA classes

PTGS : Post Transcriptional Gene Silencing (RISC)

(12)

sRNA classes

TGS : Transcriptional Gene Silencing (RITS)

siRNA

(small interferring RNA)

endo-siRNA

(endogenous)

plants fungi animals Protista

~21nt Dicer

Bi-directional or

convergent transcription mRNA base-pairing with antisense pseudogenes

TGS,

transposon regulation

hc-siRNA

(repeat-associated heterochromatic siRNA)

plants

S. pombe 24-26nt DCL3 Transposons, repeats Chromatin modification

piRNA

(Piwi-interacting RNA)

Drosophila, C. elegans, mammals, Danio rerio

24–30nt Dicer-

independent Long primary transcripts transposon regulation

heterochromatin formation, DNA methylation, transposon silencing

RNA Pol IV-V and RDRP- DNA/RNA dep RNA Pol involved in siRNA

production and amplification (plants, worms)

(13)

DICER and AGO:

two major players

PAZ

RIII

RIII

RNase III domains endonukleolytic activity

PAZ – dsRBP domain (binding dsRNA)

interacts with dsRNA 3’ end

DICER or DICER-like (DCL)

Liu and Paroo, Annu. Rev. Biochem., 2010

sRNA size determined by the distance between PAZ and RIII domains

dsRNA

siRNA

RISC

AGO

(14)

DICER and AGO:

two major players

Liu and Paroo, Annu. Rev. Biochem., 2010

ARGONAUTE

PIWI - RNAse H like domain SLICER activity in some AGOs

MID – interacts with sRNA 5’ end

PAZ – interacts

with sRNA 3’ end

(15)

Species

AGO PIWI Dicer-like RdRP

Plants A. thaliana 10 - 4 (DCL 1-4) 6

O. sativa 18 - 5 5

Fungi

S. cerevisiae - - - -

S. pombe 1 - 1 1

N. crassa 1 - 1 3

A. nidulans 1 - 1 2

Metazoa

C. elegans 5 3 2 (Dicer + Drosha) 4 D. melanogaster 2 3 3 (2 Dicers +

Drosha) -

D. rerio 4 4 2 (Dicer + Drosha) -

H. sapiens 4 4 2 (Dicer + Drosha) -

Multiple DICERs and AGOs

AtDCL2 - 4 siRNA AtDCL1 miRNA

DCL1 → 21nt miRNA → AGO1/7/10 DCL2 → 22nt siRNA

DCL3 → 24nt siRNA → AGO4/6 DCL4 → 21nt siRNA → AGO1

tasiRNA

sRNA sorting

Arabidopsis thaliana

Drosophila melanogaster

Czech and Hannon, Nat. Rev. Genet., 2010

(16)

siRNAs

Anti-viral defense

VIGS – viral induced gene silencing

endo-siRNA:

silencing of trasponsons and repetitive sequences

epigenetic silencing exo-siRNA:

transgene silencing

viral ssRNA

viral RdRP

AGO1/2 21-22nt siRNA

AGO1/2 silencing of viral

replication viral dsRNA

plants

metazoans

AGO1/2 21-22nt

siRNA mRNA cleavage

exogenous dsRNA

AGO1/2

RdRP siRNA amplification

secondary siRNA AGO

(17)

1°siRNA 1°siRNA

2°siRNA

2°siRNA

Ghildiyal and Zamore, Nat. Rev. Genet., 2009

Signal amplification

(18)

siRNA/RISC recruit chromatin modifying enzymes (DNA and histones) to DNA loci

siRNAs diced from dsRNA by DCL3 associate with AGO4

RNA-dependent RNA

polymerase (RdRP-RDR2)

siRNA signal amplification plant specific

RNA polymerase IV participates in siRNA biogenesis

ncRNA transcripts of RNA polymerase V

recruitment and targeting of silencing machinery (modifying enzymes DRM1/DRM2) to respective DNA regions

siRNAs in TGS (plants)

siRNA RDR2

2°siRNA

(19)

Pol IV/Pol V (plants)

Wierzbicki, Current Opinion Plant Biol., 2012

• lncRNA precursors to siRNA generated by Pol IV recruited by SHH1, CLSY1 provides processivity

dsRNA precursors produced by PolII

(overlapping or inverted repeat transcripts)

• RDR2 co-transcriptionally converts lncRNAs into dsRNAs

24-nt siRNAs produced by DCL3 (Dicer)

Pol V synthesizes scaffold transcripts

RDM1, DRD1, DMS3, DMS11 provide processivity

• Pol V transcripts bind AGO4/siRNA, SPT5L, IDN2

AGO4/siRNA recognizes genomic targets by basepairing with lncRNA (PolV)

AGO4 interacts with WG/GW domains of Pol IV and SPT5L

transcriptional silencing is established by de

novo DNA methylation by DRM2

(20)

Law and Jacobsen , Nat Rev Genet, 2010

chromatin remodelling factor

chromatin remodelling factor

elongation factor RNA-binding

protein

siRNAs in TGS (plants)

DNA methylation

(21)

Shiv et al., Nature., 2007

siRNAs in TGS (S. pombe)

• Pol II transcribes heterochromatin

• dsRNA generated by Rdp1 (RDRC- RNA-dep RNA pol complex) and converted to siRNA by Dcr1 (Dicer)

• siRNA loaded onto Ago1/RITS

• RITS recruits H3K9 histone methyltransferase Clr4 via Stc1

• H3K9me recruits chromatin remodellers, Chp1, Chp2, Swi6, and effectors

(SHREC, SNF2- and histone deacetylase-containing repressor complex) to facilitate

heterochromatin spreading (nucleosome repositioning)

(22)

miRNAs

Gangaraju and Lin, Nat. Rev. Mol.Cel. Biol., 2009

Voinnet, Cell, 2009; Ghildiyal and Zamore, Nat. Rev. Genet., 2009

Ago-associated 20-24 nts Dicer-dep., dsRNAs

Ago-associated 21 nts, dsRNAs plant specific miRNA-dep.

tasiRNAs…

siRNAs

Piwi-associated 26-32 nts, ssRNAs germline specific Dicer-indep.

piRNAs

(23)

miRNA BIOGENESIS (animals)

Krol et al., Nat.Rev.Genet., 2010

(24)

Jones-Rhoades et al., Ann.Rev.Plant.Biol.., 2006

miRNA BIOGENESIS

(plants)

(25)

siRNA/miRNA-dependent RNA decay

stress, development

si/miRNP

target mRNA for Slicer/Ago2 mediated cleavage and

degradation OR

repress ribosomes during elongation

promote deadenylation, decapping and mRNA degradation

inhibit translation initiation by competing for cap and/or EIF4E

Houseley and Tollervey, Cell, 2009

(26)

Huntzinger and Izaurralde, Nat. Rev. Genet., 2011

MECHANISM of miRNA-mediated PTGS (plants)

Slicer

(27)

MECHANISM of miRNA-mediated PTGS (animals)

Huntzinger and Izaurralde, Nat. Rev. Genet., 2011

(28)

TRANSLATIONAL REPRESSION

Fabian et al., Annu.Rev.Biochem., 2010

(29)

Step 1. Initial effect of miRNAs: inhibition of translation at the initiation step without mRNA decay.

Step 2. mRNA deadenylation by PAN2–PAN3 and CCR4–NOT complexes recruited by

miRISC as a consequence of translation inhibition that makes poly(A) tail more accessible.

Step3. Stimulated deadenylation potentiates the effect on translational inhibition and leads to decay of target mRNAs through the recruitment of the decapping machinery

Destabilization of target mRNA is the predominant reason for reduced protein output.

(30)

hAgo2-myc GW182 overlay

Liu et al, Nature Cell Biol, 2005

P- body proteins, DCP and GW182, colocalize with RISC components and miRNA targets

Parker and Seth, Mol. Cell, 2007

miRNA

P bodies, GW bodies, siRNAi bodies

GW182:

associates with Ago2 (mammals) or Ago1 (Drosophila)

• forms miRNP together with miRNA and Ago

• functions in miRNA dependent RNAi

• represses translation and promotes mRNA decay

Plant AGO7 accumulates in cytoplasmic siRNA bodies distinct from PB.

associated with ER membrane

• contain miRNAs

• colocalize with RDR6 and SGS3 (plant RNAi factors)

• colocalize with stress granules after heat shock

• required for ta-siRNA biogenesis

• sites of accumulation of mRNAs stalled during translation

Jouannet et al., EMBO J, 2012

(31)

REGULATION of miRNAs

miRNA processing

activators and repressors

Krol et al., Nat.Rev.Genet., 2010

(32)

ncRNA regulation

ncRNAs control gene expression (mRNAs) on different levels during various cellular and development processes

expression of ncRNAs itself is regulated via transcription, biogenesis, protein binding and turnover

Ding et al, TiB, 2009

LIN28/28B binds to let7 family pri-miRNAs and block their processing by Drosha in the nucleus

LIN28 binds to let7 pre-miRNAs and block their processing by Dicer in the cytoplasm

let7 miR-128 targets lin-28 mRNA 3’ UTR inhibiting LIN28 translation

(33)

miRNA degradation

3’ oligouridylation

PUP Poly(U) Polymerases TUTase Terminal Uridylyl Transferase

mature

Arabidopsis

Chlamydomonas

Krol et al., Nat.Rev.Genet, 2010; Kai and Pasquinelli, Nat.Str.Mol.Biol., 2010; Krol et al., Cell, 2010

precursors C. elegans

(34)

RNAi and EDITING

Nishikura,Ann. Rev. Biochem., 2010

(35)

Ota et al., Cell 2013

Additional function of ADAR1:

ADAR1 increases the rate of pre-miRNA cleavage by Dicer and facilitates loading of miRNA onto RISC complex

RNAi and EDITING

ADAR1 homodimer – editing

ADAR1/Dicer dimer - silencing

(36)

Xhemalce et al., Cell, 2012

miRNA regulation via modification

BCDIN3D methyltransferase phospho-dimethylates 5’ end of pre-miRNA, impairing its

processing by Dicer

Heo et al., Cell, 2012

Drosha processing of group II miRNA (let-7b) gives 1 nt 3’ overhang Oligo-uridylation in the presence of let-7 suppressor Lin28 (embryonic stem cells) by TUT4 leads to miRNA decay.

Mono-uridylation in the absence of Lin28 (somatic cells) by TUT7, 4 or 2 results in miRNA processing by Dicer

(37)

miRNA FUNCTION (plants)

(38)

miRNA FUNCTION (animals)

C. elegans development

lin-4 miRNA binding sites in 3’UTR

lin-14 mRNA lin-4 miRNA

lin-14 gene

C. elegans wild-type lin-14 expression during early development

lin-4 mutant

high lin14 expression

lin -14 ex pres sion

(39)

neuronal

differentiation muscle

differentiation lymphocyte

development

Stefani and Slack, Mol. Cell. Biol., 2008

miRNA FUNCTION (animals)

(40)

Non-canonical miRNAs

Maute et al, WIREs RNA., 2014

(41)

LONG ncRNAs

Laurent at al, TiG 2015

(42)

LONG ncRNAs

Laurent at al, TiG 2015

(43)

Mercer et al., Nat.Rev.Genet., 2007

ncRNA recruit chromatin

modifying complex to genes, resulting in H3meK27 and heterochromatin formation

ncRNAs act as repressors or enhancers of

transcription via binding to protein factors or DNA

ncRNAs mask 5’ splice site resulting in intron retention, recognition of IRE and translation

FUNCTIONS of LONG ncRNAs

(44)

FUNCTIONS of LONG ncRNAs

Chen and Carmichael, WIRERNA, 2010

(45)

FUNCTIONS of LONG ncRNAs

Wapinski and Chang, TiCS, 2011

(46)

MECHANISM of ACTION of LONG ncRNAs

Nagano and Fraser, Cell, 2011

Cotranscriptional recruitment of chromatin-modifying factors.

Nucleation of chromatin.

Dynamic assembly of nuclear structures:

paraspecles, nuclear bodies

Formation of higher-order chromatin loops

GUIDES (chromatin modifyiers)

TRX FACTORS

• SCAFFOLDS (RNP structures)

(47)

Xist ncRNA – inactivation of X chromosome (XCI)

Barr bodies:

heterochromatic condensed X chromosome

Xist (X-inactive specific transcript, 19 kb ) expressed from inactive X

wrapped around X

Tsix (40 kb) expressed from active X

Dosage compensation – one copy of X chromosome in females is epigenetically silenced (mammals)

Expression of XIST ncRNA → epigenetic changes → inactive state histone exchange from H2A to macroH2A

histone H3 methylation: positions H3K9, H3K27 histone deacetylation H4 (?)

DNA methylation following X inactivation (cellular memory)

RepA (repeat element) 1.6kb ncRNA (5’ of Xist) directly binds PRC2 (Polycomb)

Tsix - does not affect primary choice during XCI but protects active-X from silencing

links X reactivation and stem cell reprogramming

(48)

FUNCTIONS of ANTISENSE RNAs

Rosonina and Manley, Dev. Cell, 2010

FLC – Flowering Locus C

- MADS box transcription factor - major repressor of flowering in plants

- expression regulated by FLC antisense

two major forms of FLC antisense are synthesized

• regulated by alternative polyadenylation by RNA binding proteins, FPA and FCA, and CTSF factor FY

• short asFLC (3’ processing at site I) recruits histone demethylase FLD which introduces transcriptionally repressive histone modifications leading to FLC silencing

• long asFLC (3’ processing at site II) causes nucleosomal rearrangements at

the FLC promoter leading to FLC transcription

(49)

MALAT1/mascRNA

• Pol II polyadenylated transcript, a minor form of MALAT1,

precursor to mature MALAT1 and mascRNA

• Processing by RNAseP (5’) and RNaseZ (3’) releases 6.7 kb

MALAT1 and tRNA-like mascRNA, exported to

cytoplasm after addition of the CCA

Wilusz and Spector, RNA, 2010

MALAT1:

- metastasis-associated lung adenocarcinoma transcript 1 (NEAT2 in humans) - enriched in nuclear speckles

- possibly regulates alternative splicing (associates with SR proteins) mascRNA:

- present in the cytoplasm, processed from pre-MALAT1, function unknown

(50)

NEAT1 and MALAT1

NEAT1 MALAT1

paraspecles nuclear

specles

Chen and Carmichael, WIRERNA, 2010

(51)

TERRA – telomeric repeat-containing RNA

(yeast and human)

Luke and Lingner., EMBO J, 2009

• polyadenylated Pol II transcript

spans subtelomeric and telomeric regions

• a component of telomeric heterochromatin

associates with telomeres and telomere proteins (Trf1, Trf2)

• regulated by RNA surveillance ( Rat1 , Trf4, NMD factors, RNAse H)

• regulates telomerase (telomere shortening) via RNA-DNA

hybrids

acts in chromatin remodelling (development and differentiation)

affects telomere replication

upregulated in ICF patients

(Immunodeficiency, Centromeric

region instability, Facial anomalies)

(52)

Chen and Carmichael, WIRERNA, 2010

(53)

INVISIBLE RNAs

(54)

PERVASIVE TRANSCRIPTION OF THE GENOME

(1) protein-coding mRNA; (2) PROMPT - promoter upstream transcripts (short); (3) PASR- promoter-associated sRNAs (< 200 nts); (4) TSSa

transcription start site-associated RNAs (20-90 nts); (5) TASR –terminator associated sRNAs (< 200 nts); (6) PARL - promoter-associated long RNAs (> 200 nts); (7) tiRNAs - tiny transcription-initiation RNAs (18 nts)

SAGE, CAGE, GRO tags

antisense RNAs (can be long)

CUTs, SUTs - cryptic unstable or stable unannotated transcripts (200-600 nts)

Jacquier, Nat.Rev.Genet., 2009

(55)

CUTs, SUTs, XUTs, MUTs and ALL THAT JAZZ

CUT = Cryptic Unstable Transcripts SUT = Stable Unannotated Transcripts SAT = Ssu72-associated Transcripts

XUT = Xrn1-dependent UnstableTranscripts MUT = Meiotic Unstable Transcripts

NO LONGER

TRANSCRIPTIONAL NOISE

(yeast, mammals, worms, plants - all organisms?)

not visible in normal wild-type cells

• accumulate in RNA degradation mutants (EXOSOME, XRN family, TRAMP) or various metabolic conditions (aging, nutrient change, cell cycle etc)

• originate from widespread bidirectional promoters

„mRNA-like” Pol II transcripts (capped, polyadenylated)

Jacquier, Nat. Rev. Genet., 2009

(56)

Tisseur et al., Biochemie, 2011

GENOMIC ORGANIZATION of ncRNA

short

long

(57)

U1 and non-coding transcription

(58)

Wyers et al., Cell, 2005; Arigo et al., Mol.Cell, 2006a; Thiebaut et al., Mol.Cell, 2006, 2008; Houseley et al., EMBO J, 2007; Camblong et al., Cell, 2007; Thompson and Parker, Mol.Cell. Biol., 2007; Houseley et al., Mol. Cell, 2008; Vasiljeva et al., Mol.Cell, 2008; Luke et al., Mol. Cell, 2008;

Berretta et al., Gene Dev., 2008; Preker et al., Science, 2008; Seila et al., Science, 2008; Xu et al., Nature, 2009; Neil et al., Nature, 2009

ncRNA instability and their termination mode

Unstable CUTs

- are detected in TRAMP or exosome mutants

- are terminated by Nrd1/Nab3-dependent mechanism and polyadenylated by Trf4/TRAMP

- Nrd1/Nab3, TRAMP and exosome complexes interact - some CUTs (SRG1, IGS1-R) are polyadenylated by Pap1

- some CUTs are exported to the cytoplasm (XUTs) and degraded by Xrn1

- ncRNP composition is largely unknown

(59)

PHYSIOLOGICAL FUNCTIONS of CUTs

Camblong et al., Cell, 2007; Wery et al., WIREsSMB’11

Similar PHO84 silencing occurs in aging yeast

Stabilization of as CUT leads to H3K18 deacetylation by Hda1 at PHO84 promoter Regulation of gene expression via antisense RNA and epigenetic modification:

PHO84 (inorganic phosphate transporter)

(60)

PHYSIOLOGICAL FUNCTIONS of XUTs

Transcriptional silencing of the Ty1 transposon

Berretta et al., Gene Dev, 2008; Wery et al., WIREsSMB’11

directly or indirectly controlled by Set1

antisense TY1 XUT

polyadenylated Pol II transcript

antisense to TY1 promoter

degraded by cytoplasmic Xrn1

silences TY1 expression by promoting histone deacetylation and trimethylation (by Set1)

can act in-trans

(61)

Berretta and Morillon, Embo Rep. 2009

CUT transcribed in-cis, when stabilized, recruits chromatin modification enzymes (HDAC) to gene promoter

CUT transcribed from a distant locus, when stabilized, recruits chromatin modification enzymes (HTM) to inhibit transcrition

CUT ACTION in-cis or in-trans

(62)

ncRNA ACTION in-cis or in-trans

Guttman and Rinn, Nature, 2012

(63)

NOVEL ncRNAs: ceRNAs vs circRNAs

ceRNAs: competing endogenous RNAs, often antisense regulatory RNAs

circRNAs: circular RNAs, bind miRNAs and act as their antagonists, enhance cross-talk between ceRNAs

- ceRNA asRNA

stabilizes mRNA by sequestering miRNAs that target mRNA

- circRNA antisense RNAs arise by head-to- tail splicing, contain miR-responsive

elements and sequester miRNAs; often regulated via miRNAs and

degraded by Ago2 Slicer - circRNAs with distinct MREs may sequester different miRNA families

Taulliet al., Nat Str Mol Biol., 2013

(64)

Guil and Esteller, TiBS 2015

ceRNAs

circRNAs

(65)

circRNAs regulate transcription

exon-intron circRNAs (EIciRNAs) - localize in the nucleus

- associate with U1 snRNP

- enhance the expression of their parental gene in trans

Li at al., Nat Struct Mol Biol, 2015

(66)

NOVEL ncRNAs: eRNAs

eRNAs: enhancer RNAs, short (not always, up to 2 kb) ncRNAs transcribed from enhancer regions (RNA-Seq, ChIP-Seq)

2d-eRNAs: bidirectional, comparatively short, nonpolyadenylated 1d-eRNAs: unidirectional, long, polyadenylated

Natoli and Andrau, Annu Rev Genet., 2012

(67)

NOVEL ncRNAs: eRNAs, functions

Natoli and Andrau, Annu Rev Genet., 2012

(68)

NOVEL ncRNAs: eRNAs, functions

Quinn and Chang, Nat Rev Genet 2015

Chromosome looping

(69)

NOVEL ncRNAs: ciRNAs

ciRNAs: circular intronic lncRNAs, accumulate in human cells due to lariat debranching defect

Zhang et al, Mol Cell., 2013

- processing depends on GU-rich motive near 5’ splice site and branchpoint

- regulate parent gene expression by modulating elongation Pol II

activity

(70)

UNUSUAL ncRNAs: tRFs tRNA-derived RNA fragments

Thompson and Parker, Cell, 2009

Stress-induced enzymatic tRNA cleavage

(S. cerevisiae, D. melanogaster, A. thaliana, A. nidulans, human cell lines)

Ivanov et al, Mol Cell, 2011

(71)

UNUSUAL ncRNAs: tRFs tRNA-derived RNA fragments

> 17 short abundant tRFs (13-26 nts), generated by RNaseZ from mature (5’ and 3’ ends) and precursor (3’ trailer) tRNAs identified in the cytoplasm in prostate cancer cells. Lack of tRF1001 impairs cell proliferation.

Abundant Dicer-dependent tRFs (class I, from mature 3’ and 5’ ends) in HeLa moderately downregulate target genes.

Class II tRFs (from RNAseZ 3’ cleavage to Pol III termination, cytoplasmic) associate with Ago2-3. Function- regulation of silencing via differential

association with Ago proteins?

Haussecker et al., RNA, 2010 Cole et al., RNA, 2009

Lee et al., Gene Dev., 2009

(72)

Pircher et al, Mol. Cell 2014

Gebetsberger and Polacek, RNA Biol., 2013

Angiogenin-derived 5’-tiRNAs with terminal 5’-oligoG

repress translation in vitro and in vivo

• displace eIF4G/eIF4A from uncapped transcripts and eIF4F from m7G cap

trigger formation of stress granules (SGs)

• translational repressor YB-1 contributes to tiRNA-

mediated repression

18-mer ncRNA derived from TRM10 mRNA during salt stress in yeast

associates with polysomes

inhibits general translation

(73)

ncRNAs or sPEP (small peptides)

Lauressergues et al, Nature 2015

Alternative proteome - uORFs, AltORFs

- some “ncRNAs” code for

sPEP with functional potential

(74)

Non-canonical miRNAs

Maute et al, WIREs RNA., 2014

(75)

Unusual ways of ncRNAs

Quinn and Chang, Nat Rev Genet 2015

(76)

TAKE-HOME MESSAGE

ncRNAs have house-keeping (translation, splicing, protein sorting, RNA processing ) and regulatory functions

• ncRNAs affect processes such as transcription (chromatin structure), RNA stability and translation

The majority of eukaryotic genomes are transcribed giving rise to a variety of RNAs

At least some of the “invisible” transcripts in some conditions form functional ncRNAs

These usually act in transcriptional silencing in-cis or in-trans by recruiting chromatin modifying enzymes

• Defects in ncRNA level or activity correlate with

several diseases

(77)

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